Growing hydrocarbon-utilizing microorganisms

ABSTRACT

Hydrocarbon-utilizing micro-organisms are grown in increased yields by incubating them in a water-in-oil emulsion containing a hemochromogen wherein the oil is a hydrocarbon and the hemochromogen takes up and releases air thus presenting a greater amount of oxygen for utilization by the micro-organisms.

nited States Patent Gorring [54] GROWING HYDROCARBON- UTILIZINGMICROORGANISMS [52] [1.8. CI; ..l95/28 R, 195/109 [51] Int. Cl. ..Cl2d13/06 [58] Field of Search ..99/9, 14; 195/28, 109, 314,

[56] References Cited UNITED STATES PATENTS 2,183,192 141 940 Scholler195194 1 Feb. 15,1972

2,697,062 12/1954 Cramer ..l95/3 3,427,223 2/1969 Frankenfeld et al..l95/l Primary Examiner-A. Louis Monacell Assistant Examiner-Roger B.Andewelt Attorney-Oswald G. Hayes, Andrew L. Gaboriault and Mitchell G.Condos [57] ABSTRACT Hydrocarbon-utilizing micro-organisms are grown inincreased yields by incubating them in a water-in-oil emulsioncontaining a hemochromogen wherein the oil is a hydrocarbon and thehemochromogen takes up and releases air thus presenting a greater amountof oxygen for utilization by the micro-organisms.

2 Claims, 2 Drawing Figures the classification Eumycetes or true fungi,

GROWING HYDROCARBON-UTILIZING MICROORGANISMS BACKGROUND OF THEINVENTION 1. The field of the invention comprises a method forincreasing the growth of hydrocarbon-using micro-organisms.

2. It is conventional to grow micro-organisms in a culture mixturecomprising an oil-in-water emulsion, but so far as is known, they havenot been grown in a water-in-oil emulsion.

SUMMARY OF THE INVENTION The invention comprises a method for growingmicro-organisms by incubating them in a culture mixture in the form of awater-in-oil emulsion where the oil is a hydrocarbon. The mixture ismade up of aqueous mineral salt nutrient solution, a hydrocarbon as thesole source of carbon for energy and growth, and oxygen, and enoughhydrocarbon is present to form a water-in-oil emulsion.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS It should be noted, first ofall, that growing micro-organisms is of interest because many of themare capable of synthesizing proteins and other useful materials. Theseproteins are suitable for use as or in food for cattle and otheranimals, including humans, Increasing the growth rate of themicro-organisms, therefore, means increasing the production of proteins.

The invention is applicable to any aerobic microbial species which isable to utilize a hydrocarbon as the sole source of carbon for energyand growth, including hydrocarbon-utilizing species of bacteria, fungi,yeasts, and molds. Nonfastidious organisms are preferred, i.e., thosewhich will grow in simplified salts media without necessity foradditions of organic compounds. Species which are active animal or humanpathogens are excluded.

Of the bacteria, suitable genera include Pseudomonas, Bacillus,Flavobacterium, Sarcina, etc. Illustrative species of these genera areP. aeruginosa, P. oleovorans, P. putida, P. boreopolis, P. methanica, P.fluorescens, P. pyocyanea; B. aureus, B. acidi, B subtilis, B. urici, B.cereus, B. coagulanr, B. mycaides, B. circulans, B. megaterium;Flavobacterium aquatile; Sarcina alba, Sarcina luteum.

Other preferred genera are Achromobacter and Nocardia, as illustrated byspecies such as A. xerosis, A. agile, A. gutatus, A. superficialis, A.parvulus, A. cycloclastes; N. slamonicolor, N.

. asteroides, N. minimus, N. opaca, N. corallina, N. rubra, and N.

paraffinae. The genus Mycabacterium is useful, particularly such speciesas M. parafficum, M. phlei, M lacticola, M. rhodochrous, M. smegmatis,M. rubrum, M. luteum, M. album, M. byalinicum, and M. leprae.

Still other hydrocarbon-utilizing bacteria are Methanomonas melhanicaand Methanomonas sp.; Micrococcus paraffinae; B. alipharicum, B. hidium,and B. benzali from the genus Bacterium; and species of Micromonaspora.Other useful genera include Brevibacterium, Aerobacter, and Corynebarterium.

Of the fungi, the method is applicable to any fungus within butpreferably those from the class Fungi Imperfecti or from the classPhycomycetes. Preferred fungi from the class Fungi Imperfecti arespecies of the genera Aspergillus and Penicillium, as illustrated by A.niger, A. glaucus, A. oryzae, A. flavus, A. terreus, A. itaconicus; P.notatum, P. chrysogenum, P. glaucum, P. griseafulvum, P. expansum, P.digitalum, P. italicum, etc. Other suitable organisms include variousspecies of the genera Monilia, Helminthosporium, Alternaria, Fusarium,and Myrothecium. Preferred fungi of the class Phycomycetes includespecies from the genera Rhizopus and Mucar, such as R. nigricans, R.oryzae, R. delemar, R. arrhizus, R. stolonifer, R. sp.; M. mucedo, M.genevensis.

Some of the foregoing genera of fungi are also characterized as molds,such as Aspergillus, Penicillium, Rhizopus, and Mucor, but it will beunderstood that all are true fungi or Eumycetes.

Of the yeasts, the preferred organisms are of the familyCryptococcaccae, and particularly of the subfamily Cryptococcoidae.Preferred genera are T0rul0pris(or Torula) and Candida. Preferredspecies are Candida Iipulytica, Candida pulcherrima, Candida utilis,Candida ulilis Variali major, Candida tropicalis, Candida intermedia,and Torulopsis colliculasa. Other useful species are Hansenula anomala,Oia'ium lactia, and Neurospora sitophila.

The hydrocarbon is one that is in the liquid phase at incubationtemperature so as to be able to form a water-in-oil emulsion. Aliphatichydrocarbons are preferred, and these may be saturated or unsaturated,straight or branched chain hydrocarbons having up to 20 or 30 or 40 ormore carbon atoms. Saturated straight-chain hydrocarbons having up to 20carbons are particularly desirable. Cyclic hydrocarbons, comprisingaromatic and alicyclic compounds, are also of use, includingalkyl-substituted cyclic compounds having one, two, or more alkylsubstituents each of any suitable length, chain configuration, anddegree of saturation, and in which the cyclic moiety is aromatic orcycloparaffinic. Alkyl-substituted aromatic hydrocarbons includetoluene, the various xylenes, mesitylene, ethylbenzene, p-cymene, thediethylbenzenes, and the isomeric propylbenzenes, butylbenzenes,amylbenzenes, heptylbenzenes, and octylbenze nes. Among the usefulalkyl-substituted cycloparaffins are methylcylcopentane, the diandtrimethylcyclopentanes, ethylcyclopentane, the diethylcyclopentanes, thevarious propyl-, butyl-, amyl-, hexyl-, and octylcyclopentanes. Also thealkylcyclohexanes, which are substituted in a manner corresponding tothe foregoing alkylcyclopentanes, and further including such compoundsas the various tetramethylcyclohexanes, methylethylcyclohexanes,methylpropylcyclohexanes, and the like.

Crude oils, various petroleum fractions, residua, etc., are of use.

It will be appreciated that the hydrocarbon may be in the liquid phasenot only by having a suitable melting point but also by being dissolvedin a suitable solvent. The hydrocarbons contemplated in the precedingparagraphs are those which are normally liquid at incubationtemperature. However, other useful hydrocarbons are those which arenormally gaseous at incubation temperature. However, other usefulhydrocarbons are those which are normally gaseous at incubationtemperature, such as methane, ethane, propane, butane, and other C3 toC5 hydrocarbons. These gaseous hydrocarbons may be dis solved in anormally liquid hydrocarbon, such as a petroleum fraction in thegasoline or kerosene boiling range, or in an aland such ions aspotassium, magnesium, phosphate, and

sulfate, as well as ions of trace elements like molybdenum, cobalt, etc.Traces of manganese, iron, and calcium may be present. As water isincluded in the nutrient, most of these ions will usually be present insufficient quantity in ordinary potable water supplies. However, it isdesirable to add the ions to the nutrient to insure their presence insufficient quantity for growth. Usually the nutrient consists primarilyof water, which may constitute 99 percent, or more, by weight of thenutrient, although it may also constitute a lesser portion, going downto 50 percent thereof. Generally any proportion of water heretoforeemployed in microbial growth may be used. A suitable mineral saltsnutrient may be listed as follows, the components being dissolved inenough water to make 1 liter of solution:

TABLE 1 Potassium monohydrogen phosphate 60 g. Sodium dihydrogcnphosphate 9.0 Sodium molybdate 0.006 Cobaltic chloride 0.006 Magnesiumsulfate 06 Ammonium sulfate Another suitable mineral salts nutrient isas follows:

TABLE 2 Sodium monohydrogen phosphate 9 g. Potassium dihydrogenphosphate 6 Ammonium sulfate 6 Magnesium sulfate 06 Sodium carbonate 03Calcium chloride 0.03 Ferrous sulfate 0.0]5 Manganese sulfate 0.006Cobalt chloride 0.006 Sodium molybdutc 0.006

The method generally comprises incubating the micro-organism in themineral nutrient in the presence of the hydrocarbon and oxygen. Duringincubation, the culture mixture is maintained under conditions to insureoptimum growth of the microorganism. The temperature for example shouldbe maintained between about and about 55 C., preferably from 20 to 40 C.The pH is maintained near neutrality, namely, about 7.0, although it mayrange between about 5.5 and 8.5. The mixture is maintained in acondition of agitation as by shaking, or by using propellers, paddles,rockers, stirrers, or other means ordinarily employed for effectingagitation ofa liquid mixture. Also included is agitation produced bycirculating air in the mixture after introducing the same thereto bymeans ofa sparger.

The proportions of the aqueous mineral nutrient which includes water anddissolved salts) are such as to provide the water-in-oil emulsion, thehydrocarbon forming the continuous phase. The emulsion is preferablyformed and maintained by agitation of the culture mixture, and suitablymay comprise at least 66 percent, and going up to 80 or 90 percent ormore, of hydrocarbon, the balance being aqueous mineral nutrient, volumebasis. According to this preferred method of forming the emulsion, oneor more emulsifying agents are present, either being produced in theculture mixture during the fermentation, or being added per se.Emulsifying agents added per se may be of ionic or nonionic character,it being understood that they are suitable to promote a water-in-oilemulsion. A water-in-oil emulsion may be formed and maintained at lowerhydrocarbon proportions, extending from about 66 percent down to orpercent, balance aqueous mineral nutrient, volume basis, and at theseproportions it is preferred to add one or more emulsifying agents per seof the kind described. Some illustrative agents include oleates andstearates like sorbitan sesquioleate, sorbitan monooleate, sorbitanmonostearate, polyoxyethylene oxypropylene glucoside oleate, etc.Preferably the agents have an HLB (Hydrophile- Lipophile Balance) ofabout 3 to 6. (Numerical HLB values are explained in a publication ofAtlas Chemical Co. entitled Atlas Surfactants," where they are shown torange from about 1 to 30, with the lower values indicating a morelipophilic material and the higher values indicating a more hydrophilicmaterial.) Agents of suitable HLB value may also be obtained by blendingtwo or more other agents. The useful agents are used in conventionalamounts, and preferably they are edible or nontoxic.

The emulsion may be conveniently formed within the fermentor, althoughit is also feasible to prepare it by means ofa conventional homogenizerand then add it to the fermentor.

The fermentor may be open to the atmosphere, and with agitation ofthemixture, the surface thereof exposed to the atmosphere is continuouslyrenewed and oxygen is taken up from the atmosphere. It is preferred,however, to keep the fermentor closed and to supply oxygen by bubblingair through the mixture, thereby also providing desired agitation.

In connection with the supply of oxygen to the fermentor, upon which thecell growth rate depends, the invention provides a marked advantage overconventional cell production using a culture mixture comprising anoil-in-water emulsion, In the conventional method, oxygen is transferredfrom air bubbles through the continuous aqueous phase of the oil-inwateremulsion to the growing cells disposed in such phase, and this transferrate is dependent on the solubility of oxygen in the aqueous phase. At37 C., a conventional incubation temperature, the solubility of oxygenin the aqueous phase. under equilibrium conditions, is about 6 ppm Inthe present method, oxygen is transferred from the air bubbles throughthe continuous hydrocarbon phase of the water-in-oil emulsion at a rate,under equilibrium conditions, corresponding to its solubility in suchphase, which ranges from 60 to lOO ppm, and from the hydrocarbon phasethe oxygen is transferred to the cells in the dispersed aqueous phase.It will thus be seen that the transfer rate through the continuoushydrocarbon phase is l0 to 16 times greater than that through thecontinuous aqueous phase; and the cell growth rate in the water-in-oilemulsion is greater than that in the oil-in-water emulsion by at leastan order of magnitude.

A particularly suitable method for growing cells in the water-in-oilemulsion, while supplying air in amounts adequate not only for growthbut also to provide agitation, is shown in connection with the apparatusdiagrammatically illustrated in FIGS. 1 and 2, the latter figure being asection along the line 2-2 of FIG. 1. The method involves an airliftoperation carried out in a U-shaped device 10 comprising a pair ofcolumns 11, 12 each connected at the bottom by a U- bend l3 and eachopen at the top. The open tops 14, 15 are enclosed within a cylindricalportion 16 having a cover 17 over its open upper end. The lower end 18ofcylinder I6 is cut at a slant and closed off by the slanting wall 19.The upper end of column 11 is also cut at a slant, and the opening 14therein lies in the plane of the slanted wall 19. Thus, liquid incylinder 16 drains through such open end 14 into column 11. Column 12extends into cylinder 16 for some distance, terminating in a flared endportion 20. In normal operation, liquid fills cylinder 16 to a level asindicated at 21, and into such liquid there extend a pl-l-indicatingcontrol 22 and a temperature-indicating device 23. A vent at 24 allowsgases to pass out.

The columns 11 and 12 are constructed of sections, as shown, althoughany other suitable construction may be employed. Between each adjacentpair of sections there are provided watertight gaskets. An air inlettube 25 conducts air from a source not shown to a sparger device 26located below the middle of column 12. At 27 is a valved drain tube forremoving samples.

While various materials may be used in the construction of the device,suitably the columns may be of glass, the cylinder of metal, the coverof transparent material, and the gaskets of Teflon plastic.

The device may be operated by removing cover 17 and charging into theflared end 20 of column 12, in desired proportions, the aqueous mineralnutrient and the hydrocarbon. Enough material is added to fill column11, column 12 to the level indicated at 21, and the cylinder 16 to saidlevel. A micro-organism is also present, being added to the materialeither before or after charging of the same to the device. Air is passedinto column 12 through the sparger at a desired rate, this being atleast sufficient to move the liquid upwardly in column 12, over the top15 thereof, and into the lower portion of cylinder 16. A flow is thuscreated, note the arrows, with liquid in column 11 moving over intocolumn 12, and liquid in the cylinder descending by gravity into column11. The speed of this flow may be estimated visually and controlled byadjusting the air rate.

The culture mixture in the device may be heated in any suitable way, asby inserting an electrically heated rod or other device (not shown) intothe liquid in cylinder 16, or in any other desired way. Automatictemperature control means, of conventional kind, may be used to maintaina constant temperature. Control over pH is provided by the instrument22, and if necessary, suitable adjustments are made from time to time byappropriate addition of an alkaline or an acid material. Air collectingin cylinder 16 is vented through line 24 along with any other gases.

It will be apparent that the airstream introduced through line 25 notonly brings oxygen into the system but also provides for the agitationof the mixture and its movement from one column to the other. Suchagitation and movement make for good contact between the cells and theother components of the mixture. A typical airflow rate is 0.] cu. ft.per minute per liter of liquid at standard conditions, although the ratemay be increased to 5, or more times this value or decreased to or 10percent, or less, ofsuch value,

Operation of the foregoing method may be on a batch or a continuousbasis. In the former, the initial charge of culture mixture ismaintained throughout, there being no additions and, except for testsamples, no withdrawals. In the latter, additions and withdrawals aremade, either continuously or intermittently, throughout the course of arun.

In a batch run, circulation of the mixture between columns 11 and 12 maybe carried on as long as desired, but usually it is brought to a haltwhen there is no further increase in cell growth. Growth may be followedby removing test samples of the mixture from time to time through line27 and analyzing the same as by means of optical density measurements,as

described below in Example 1. When it is decided to stop thefermentation, the airflow and the heat input are shut off, and themixture is drained from the system through line 27 to a settling tankwhere separation of phases is brought about by allowing the mixture tostand, although other separation means may be employed. One of theadvantages of the invention is that phase separation, as brought aboutby standing, can be achieved almost spontaneously, or in a matter ofseconds, and generally in less than a minute. The oil phase rises to thetop of the settling tank, is removed, and held for reuse in another run.The aqueous phase, in which the cells collect, is subjected to asuitable separation step, such as filtration, centrifugation, settling,etc., whereby the cells are recovered. The resulting used aqueousmineral nutrient may, if desired, be worked up to recover any chemicalvalues, and if this is not feasible or desired, it may be discarded, orif justified it may be reused.

Cell yields of up to 20 g./l. of culture mixture, or more, areobtainable. By comparison, a yield of no more than about 5 g./l. isobtainable using a culture mixture in the form of an oilin-wateremulsion.

In a continuous run the procedures used in a batch run are applicableexcept that additions of hydrocarbon and aqueous mineral nutrient, andwithdrawals of culture mixture, are made throughout the course of therun either on an intermittent or a continuous basis. These additions andwithdrawals are balanced not only to maintain the quantity or level ofculture mixture in the apparatus but also to maintain the condition ofthe emulsion, i.e., as a water-in-oil emulsion of substantially constantcomposition. The withdrawn culture mixture may be continuously separatedinto phases, the oil phase reused as by recycling, and the cells removedand recovered from the aqueous phase. Used aqueous phase may be reusedas is, or it may first be reconstituted by addition of mineral salts andthen reused; or, if its condition is deemed unsuitable, it may bepurified by removing toxic products or it may be discarded. If desired,some of the product cells may be recycled, either before or afterseparation from the aqueous phase, as such recycling may provide somestimulation for further growth. The withdrawals and additions may beinitiated at any desired time, either at the point of maximumconcentration or growth of the cells or at some earlier point;preferably they are initiated at a point subsequent to the start of thefermentation. The point of maximum growth may easily be established asby plotting a graph of variance between cell concentration and time ofrun. An advantage of a continuous run is to provide a continuous yieldof cells from a given apparatus. Another advantage is that toxicproducts, i.e., products toxic to the cells, which tend to collect inthe aqueous phase, may be continuously removed from the system.

' It is feasible to add to the culture mixture a substance which takesup and releases oxygen, preferably one that is superior in this respectthan the hydrocarbon. Such substance or com pound can then take upoxygen from the introduced air and at some subsequent point in the flowof the culture mixture release at least a part of the oxygen to thecells. In this way a greater amount of oxygen may be presented to thecells for utilization by them. A compound of interest for this purposeis hemoglobin, a water-soluble material comprising a complex of aniron-containing pigment portion, called heme or hemin, with a proteincalled globin. Hemoglobin has the ability to combine loosely withmolecular oxygen, forming oxyhemoglobin, but the union is loose and canbe dissociated under relatively simple conditions, such as a change ofpartial pressure of oxygen or of pH, forming oxygen and reducedhemoglobin. Thus, the hemoglobin can pick up oxygen in a region ofhigher oxygen partial pressure and release it in a region of loweroxygen partial pressure. Such regions may be identified in the apparatusof FIG. 1, the former being adjacent and just above the introduced airin column 12 and the latter being more remote from such airstream as incolumn 11.

The hemoglobin complex may be separated into heme and globin. The hemeis capable of combining with various other proteins, like albumin, andwith bases like pyridine, nicotine, ammonia, etc., yieldinghemopyridine, hemonicotine, etc. Collectively such compounds are calledhemochromogens. They react with oxygen similarly to hemoglobin, and areof similar use. It may be noted that hemoglobin itself is ahemochromogen. Hemoglobin is found in the blood of all vertebrates. Hemeis even more widely distributed in nature and may be used to form acomplex with a desired nitrogen-containing base or a protein.

The invention may be illustrated by the following examples.

EXAMPLE 1 In a batch run, a culture mixture was prepared comprising 6.5liters n-hexadecane, 3.0 liters of fresh aqueous mineral nutrient of thecomposition set forth in Table l, and 250 mg. Brevi-bacterium sp. Themixture had a pH of 7.0. It was charged to an airlift fermentor, of thetype illustrated, of about 10 liters capacity, heated to 36 C., and airbubbled through at a rate of about 1.8 cu. ft./min. As growth tookplace, the pH of the mixture fell to 6.0, at which point it wasmaintained by addition of ammonium hydroxide. Distilled water andn-hexadecane were added periodically to replace losses by evaporation.

The concentration of the cells was measured periodically by means ofoptical density determinations made on total or complete samples ofculture mixture. One cc. of fermentor sample was diluted in 8 to 10 cc.of 2-propanol, centrifuged, then redispersed in water in preparation fora density measurement. Centrifugation of each sample showed the liquidphase of the same to be composed of about oil and /3 water or aqueousmedium. Staining with a water-soluble dye (Nigrosin) revealed that theliquid phase was a water-in-oil emulsion.

At all cell concentrations it was found that the aqueous phase of thewithdrawn samples was able to separate from the hydrocarbon phase inabout 20 seconds, a phenomenon not observable in the case of a culturemixture comprising an oilin-water emulsion. Owing to this rapidseparation of phases, a foam-free aqueous phase was formed whichcontained most of the cells; and over it was a hydrocarbon phase. Afterabout 10 minutes, a distinct settling-separation of the cells in theaqueous phase was noticed, with no dispersed oil droplets present.Optical density was measured by testing samples of culture mixture forthe adsorption of visible light rays of a wavelength of 400 millimicrons(0.4 micron) in a Bausch and Lomb colorimeter. The following table showsthe increase in cell growth at periodic intervals throughout the run.

It is apparent that cell growth increased continuously throughout therun. Starting at about the 7th hour and continuing to about the 13thhour, the increased growth was sharp, the concentration increasing from1.3 to 7.8 g./l. If the data is plotted to give a concentration-vs.-timecurve, it will be seen that this sharp increase corresponds to an almostvertical portion of the curve. From the 13th to the 25th hours, growthlevelled off but was still continuous. A high of 8.4 g./l. was reached.

EXAMPLE 2 In another run carried out in substantially the same manner asin the preceding example, the yield of cells reached a maximum of about20 g./l. it is felt that in this run contamination of the culturemixture by toxic products was probably less a factor than in thepreceding run.

It will be understood that the invention is capable ofobvious variationswithout departing from its scope.

In the light of the foregoing description. the following is claimed.

1. Method for increasing the growth of cells of a hydrocarbon-utilizingmicro-organism comprising incubating said micro-organism in a mixturecomprising an aqueous mineral salt solution and a hydrocarbon as thesole source of carbon for energy and growth of said micro-organism,forming said mixture into a water-in-oil emulsion where the oil is saidhydrocarbon, introducing an oxygen-containing gas into said mixture,introducing a hemochromogen into said mixture, growing said cells insaid mixture, then separating said mixture into an oil phase and anaqueous phase containing most of said cells, and recovering said cellsfrom said aqueous phases.

2. Method for increasing the growth of cells of a hydrocarbon-utilizingmicro-organism which comprises incubating said micro-organism in amixture comprising an aqueous mineral salt solution and a hydrocarbon asthe sole source of carbon for energy and growth of said micro-organism,said hydrocarbon comprising at least 35 percent by volume of saidmixture so that the latter forms a water-in-oil emulsion, introducing anoxygen-containing gas into said mixture, introducing a hemochromogeninto said mixture, growing said cells in said mixture, then separatingsaid mixture into an oil phase and an aqueous phase containing saidcells, recovering said cells from said aqueous phase, and in this wayproducing a larger yield of said cells than in a like method whichhowever employs said mixture in the form of an oil-in-water emulsion.

UNITED STATES PATEN? OFFICE CERTIFICATE OF C OR RECTEON Patent No. 3,642 ,577 Dated February 15 1972 Inventofls) Robert L. Gorring It iscertified that error appears in the above-identified patent and thatsaid Letters Patent are hereby corrected as shown below:

On the title page, under list of attorneys, add the name --Frederick E.Dumoulin--.

Column 1, line 46, "N. slamonicolor" should be -N. sa Imon1coIor--. JColumn 3, Table 2, first Iine, "9 g." should be --9 g.jl.

Column 8, last line of claiml, "phases" should be --pha se--.

Signed and sealed this 18th day of July 1972.

(SEAL) Attest: A

EDWARD M.F'LETCHER JR. ROBERT GOT'I'SGHALK Attesting OfficerCommissioner of Patents FORM PC4050 (169) uscoMM-oc 60376-P69 Q U.S.GOVERNMENT PRlNTING OFFICE: I959 0356-334

2. Method for increasing the growth of cells of a hydrocarbon-utilizingmicro-organism which comprises incubating said micro-organism in amixture comprising an aqueous mineral salt solution and a hydrocarbon asthe sole source of carbon for energy and growth of said micro-organism,said hydrocarbon comprising at least 35 percent by volume of saidmixture so that the latter forms a water-in-oil emulsion, introducing anoxygen-containing gas into said mixture, introducing a hemochromogeninto said mixture, growing said cells in said mixture, then separatingsaid mixture into an oil phase and an aqueous phase containing saidcells, recovering said cells from said aqueous phase, and in this wayproducing a larger yield of said cells than in a like method whichhowever employs said mixture in the form of an oil-in-water emulsion.